Confocal microscopy and the H⁺-sensitive fluorophore carboxyseminaphthorhodafluor-1 (SNARF-1) were used to measure either intracellular pH (pH_i) or extracellular pH (pH_o) in isolated, arterially perfused rabbit papillary muscles. Single-excitation, dual-emission fluorescent images of the endocardial surface and underlying myocardium to a depth of 300 µm were simultaneously recorded from perfused cylindrical muscles suspended in a controlled atmosphere oriented oblique to the focal plane. Contraction was inhibited by the addition of butanedione monoxime. In separate muscles, pH_o was measured during continuous perfusion of SNARF-1 free acid. pH_i measurements were made after the muscle was loaded with SNARF-1/AM and the extracellular space was cleared of residual fluorophore. Initial experiments demonstrated the uniformity of ratiometric measurements as a function of pH, image depth, and fluorophore concentration, thereby establishing the potential feasibility of this method for quantitative intramural pH measurements. In subsequent experiments, the method was validated in isolated, arterially perfused rabbit papillary muscle during normal arterial perfusion and as pH_i and pH_o were altered by applying CO₂ externally, exchanging HEPES and bicarbonate buffers, and changing pH_i with NH₄Cl washout. We conclude that in situ confocal fluorescent microscopy can measure pH_i and pH_o changes at the endocardial surface and deeper endocardial layers in arterially perfused ventricular myocardium. This method has the potential to study pH_i regulation in perfused myocardium at boundaries where diffusion of gases, metabolites, and peptides are expected to modify processes that regulate pH_i.